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Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Ilsley, Melissa LU ; Capellera-Garcia, Sandra LU ; Dhulipala, Kishori; Johansson, Alban and Flygare, Johan LU (2018) In Journal of visualized experiments : JoVE
Abstract

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here... (More)

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of visualized experiments : JoVE
issue
142
publisher
JoVE
external identifiers
  • scopus:85059265110
ISSN
1940-087X
DOI
10.3791/58464
language
English
LU publication?
yes
id
4079003c-61fa-4b68-851a-36488b4bbaf6
date added to LUP
2019-01-11 14:10:01
date last changed
2019-01-11 14:10:01
@article{4079003c-61fa-4b68-851a-36488b4bbaf6,
  abstract     = {<p>Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.</p>},
  articleno    = {e58464},
  author       = {Ilsley, Melissa and Capellera-Garcia, Sandra and Dhulipala, Kishori and Johansson, Alban and Flygare, Johan},
  issn         = {1940-087X},
  language     = {eng},
  month        = {12},
  number       = {142},
  publisher    = {JoVE},
  series       = {Journal of visualized experiments : JoVE},
  title        = {Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors},
  url          = {http://dx.doi.org/10.3791/58464},
  year         = {2018},
}